Quorum Sensing

9. MeasureA 600 and LCPS, simultaneously (seeNote 2). Average
   theA 600 and LCPS measurements of the replicates. Normalize
   the averaged LCPS to the averagedA 600 to obtain PA14-R3
   reporter activity. Extrapolate 3OC 12 -HSL concentration in the
   treated and untreated supernatants based on the values
   obtained for the calibration curve (seeNote 6).

3.5 Elastase Assay
Procedure

1. Set up 1.5 ml tubes each one containing 20 mg of Elastin-
   Congo Red and 1 ml of Protease Buffer (seeNote 7).


2. Add 100μl of culture supernatant collected in Subheading3.4,
   step 3(seeNotes 5and 8 ) to the tube containing the Elastin-
   Congo Red suspension. Prepare a control sample (blank) by
   adding 100μl of sterile LB instead of the culture supernatant.


3. Incubate 2 h with gentle shacking at 37C.


4. Centrifuge for 5 min at 11,000gat room temperature.


5. Measure absorbance at 495 nm wavelength (A 495 ) of the clear
   supernatants in plastic microcuvettes, using as blank the con-
   trol sample (see above). Normalize with respect to theA 600 of
   the corresponding culture measured in Subheading3.4,step 3.

3.6 Pyocyanin Assay
Procedure

1. Add 3 ml of chloroform to 15 ml conical tubes containing 5 ml
   of the supernatants collected in Subheading3.4,step 3(see
   Notes 5and 8 ). Mix vigorously by vortexing for 10 s. As
   control sample (blank), use 5 ml of sterile LB in place of the
   bacterial supernatant.


2. Centrifuge the tubes at 3,000gfor 5 min.


3. Transfer 2 ml of the lower organic phase in clean 15 ml conical
   tubes (the lower organic phase is blue if pyocyanin is present)
   and add 1 ml of 0.2 N HCl. Mix vigorously by vortexing for
   10 s.


4. Centrifuge the tubes at 3,000gfor 5 min.


5. Transfer 800μl of the upper aqueous phase in plastic micro-
   cuvettes (the upper aqueous phase is pink if pyocyanin is
   present).


6. Measure the absorbance at 520 nm wavelength (A 520 ), using as
   blank the control sample (see above). Normalize with respect
   to theA 600 of the corresponding culture measured in Subhead-
   ing3.4,step 3.

4 Notes

1. By using bacterial biosensors in which light emission is induced
   by exogenous C 4 -HSL, PQS, or other QS molecules different
   from 3OC 12 -HSL, similar coculture-based approaches can be
   designed to identify inhibitors of other QS systems. Please

A Coculture-Based Approach to Identify QS Inhibitors 293